Compounds are described which represent novel, efficacious, and less toxic alternatives to current antiparasitic/antifungal treatments. Compounds having action via the biochemical mechanism of inhibition of lipid synthesis and/or metabolism and/or excretion, either by direct or indirect inhibition, will have either singly or in combination antiparasite/antifungal activity. Such compounds, in most cases, are not chemically related by structure or chemical class to each other. The compounds are identified as antiparasitics and/or antifungals based on mechanism of physiologic action. Data supporting xe2x80x9cnovel usexe2x80x9d as antiparasite/antifungal compounds are given. Many compounds herein described are FDA-approved and marketed for human use for nonparasitic/nonfungal indications. Thus, the human pharmacokinetics for oral absorption, elimination rates/mechanisms, and dose-related toxicity are known.
Status of Leishmaniasis, trypanosomiasis, and trichomoniasis
Current drugs most frequently used to treat leishmaniasis all require parenteral administration, date back 40xe2x86x9250 years, and all have such severe side-effects that treatment only in a hospital setting is recommended (Bryceson, 1968, East African Med J 45, 110-117; Bryceson, A., 1987, The Leishmaniases in Biology and Medicine, Vol II Clinical Aspects and Control, Academic Press, New York, pp. 847-907). No antileishmanial is Food and Drug Administration (FDA) approved and there is no chemoprophylaxis for any leishmanial disease. Topical treatment for leishmanial disease is not effective even for cutaneous disease forms because leishmaniasis is a systemic disease (Neva, et al., 1997, Trans R Soc Trop Med Hyg 91, 473-475). There is no general vaccine for leishmaniases, although a live vaccine is used in the Middle East for certain Leishmania (Leishmania) tropica/Leishmania (Leishmania) major to prevent facial scarring. Drug resistance is so severe in certain endemic regions that thousands are dying in India of untreatable, multidrug resistant visceral leishmaniasis; and in Northern Africa as a result of malnutrition exacerbated disease (Cerf, et al., 1987, J Inf Dis 156, 1030-1033; de Beer, et al., 1991, Am J Trop Med Hyg 44, 283-289; Sundar, 1997, Acta Parasitol Turicica 21, suppl 1, 128).
Immunodeficiency, either as the result of leishmanial tubercular- or HIV coinfections, poses serious therapeutic difficulties as leishmanial coinfection is reported to potentiate the pathology of both these bacterial and viral infections (Alvar, et al., 1997, Clin Microbiol Rev 10, 298-319; Bernier R, et al., 1995, J Virol 69, 7282-7285; Bryceson, 1987, supra; Faraut-Gamarelli, et. al., 1997, Antimicrob Agents Chemother 41, 827-830). Global travel and commerce result in patients having complex disease exposure history, and transportation of leishmanial parasites far from their anticipated endemic regions making both diagnosis and patient management difficult (Albrecht, et al., 1996, Arch Pathol Lab Med 120, 189-198). Leishmaniases have an annual incidence of 2-3 million new cases per year with 12 million infected and 350 million at risk in 88 countries worldwide (Croft, 1988, Trends Pharmacol Sci 9, 376-381; World Report on Tropical Diseases, 1990). The need for a orally administered antileishmanial of low toxicity is critical.
Two major groups of diseases caused by flagellate protozoa are African sleeping sickness (Trypanosoma brucei spp.) and trichomoniasis (Trichomonas/Tritrichomonas) exhibited as trichomoniasis vaginalis and trichomoniasis foetus.
African trypanosomiasis affects both domestic and wild animals as well as humans in mainly rural settings (Kuzoe, 1993, Acta Tropica 54, 153-162; World Health Organization (WHO), 1995, Tropical Disease Research, Twelfth Programme Report, Geneva Switzerland) while trichomoniasis is a cosmopolitan disease in men as well as women, and a threat to cattle breeding in most agricultural areas of the world (Hammill, 1989, Obstet Gynecol Clin North Am 16, 531-540; Levine, 1985, Veterinary Protozoology. Iowa State Univ. Press, Ames, pp 59-79). Treatment of the organisms causing these diseases presents problems, in part, due to the toxicity of existing agents, and the development of resistance to existing drugs (Kuzoe, 1993, supra; Lossick, 1989, Trichomonads Parasite in Humans. Springer-Verlag, New York, pp 324-341).
African trypanosomiasis is endemic in over 10 million square kilometers of sub-Saharan Africa, affecting humans and all domesticated livestock (WHO, 1995, supra). There are an estimated 25,000 new cases of human disease yearly and an animal incidence of 250-300,000 cases but these estimates are low, based on recent civil unrest and lapses in local tsetse fly control and medical surveillance (WHO, 1995, supra). The primary drugs for human and veterinary trypanosomiasis have been in use for  greater than 50 years. Resistance is spreading, especially to the only available agent for late stage central nervous system (CNS) human disease, melarsoprol (van Nieuwenhove, 1992, Ann Soc Belg Med Trop 72, 39-51; Kuzoe, 1993, supra). Melarsoprol is also toxic, with a 3-5% incidence of cerebral episodes reported (Pepin and Milord 1994, Adv Parasitol 33, 2-47; Wery, 1994, Int J Antimicrob Agents 4, 227-238). Veterinary trypanocides include diminazene (Berenil(copyright)) and isometamidium (Samorin(copyright)) which are used prophylactically for control of disease in cattle herds (WHO, 1995, supra; Kaminsky et al., 1993, Acta Tropica 54, 19-30). Resistance to both agents has been documented in field studies (Kuzoe, 1993, supra; Schoenfeld et al., 1987, Trop Med Parasitol 38, 117-180; Williamson, 1970, The African Typanosomiases. Allen and Unwin, London, pp 125-224). For these reasons, there is an urgent need to develop new trypanocides.
Trichomonas vaginalis is one of the most prevalent sexually transmitted pathogen of the human urogenital tract. It infects the vaginal epithelium, causing severe irritation and the development of a discharge. In addition to social distress caused by the disease, recent evidence suggests a high incidence rate between cervical cancer and trichomoniasis (Gram et al., 1992, Cancer Causes and Control 3, 231-236). The disease is widespread, with about 3 million cases in women annually in the United States alone (Hammill, 1989, supra). Chemotherapy for human trichomoniasis relies on a group of 5xe2x80x2-nitroimidazoles, with metronidazole (Flagyl(copyright)) being the most utilized. In the United States, metronidazole is the only available agent, although other derivatives are used in Europe and other areas. Since metronidazole has been in continuous use since 1955, there has been increasing reports of metronidazole-resistant vaginitis (Meingassner and Thurner, 1979, Antimicrob Agents Chemother 15, 254-258; Wong et al., 1990, Australia-New Zealand J Obstet Gynecol 30, 169-171; Voolman and Boreham, 1993, Med J Australia 159, 490). Because of its potential to produce free radicals upon reduction, it is potentially mutagenic and not given to pregnant women (Lossick, 1989, supra). At present, there is no alternative to the 5xe2x80x2-nitroimidazoles for therapy of metronidazole-refractory disease, nor for treatment of pregnant women.
Trichomonas foetus is the agent of bovine trichomoniasis, causing reproductive failure. Parasites are spread by infected bulls, multiply in the vagina and invade the cervix and uterus. One to 16 weeks after breeding, abortion of the fetus occurs (Levine, 1985, supra). If the placenta and fetal membranes are eliminated following abortion, the cow may spontaneously recover. If some of these tissues remain inside the animals, permanent sterility may result. There is no satisfactory treatment for diseased cows, while treatment of bulls is tedious and expensive. Aminoquinuride (Surfen(copyright)) or acriflavine (Trypaflavine(copyright)) may be used topically, with dimetridazole injected into the urethra. Unless the bull is valuable, it is usually destroyed (Levine, 1985, supra). The disease is common in open range breeding ranches and may reach epidemic levels. In Australia, 40-65% of cattle were reported to be infected, while the prevalence in California was reported to be 14% (Yule et al., 1989, Parasitol Today 5, 373-377). The economic losses due to bovine trichomoniasis have been estimated to be $665/infected dairy cow, while the widespread prevalence of the disease would account for tens of millions of dollars annually (Yule et al., 1989, Parasitol Today 5, 373-377). The overall situation for chemotherapy of trichomoniasis therefore, is the reliance on a single drug as drug class for chemotherapy of human disease, and no effective control measures for bovine trichomoniasis.
Preliminary evidence from our ethnomedical and ethnobotanical drug discovery research as well as background literature describing different aspects of the parasite""s sterol pathway and cholesterol requirements and importance to parasite survival, has led to the discovery of compounds chosen on the basis of their physiological function on different parts of the sterol synthesis, and/or excretion, and/or metabolism which offer potential chemotherapeutic target(s) having low toxic potential for man. Several of these compounds have been tested for their antiparasitic/antifungal activity as described in the Examples.
The following is a brief summary of the background and data which led to the discovery of the antiparasitic/antifungal compounds of the present invention.
Lipids comprise up to 15% of the total dry weight of Leishmania spp. (Meyer and Holz, 1966, J Biol Chem 241, 5000-5007; Beach, et al., 1979, J Parasitol 65, 203-216; Fish, et al., 1981, Mol Biochem Parasitol 3, 103-116). Lipid metabolism is critical to parasite membrane transport, cell replication, and, therefore, to survival. The lipid metabolism of Leishmania spp. including precursors, synthetic pathways, regulator molecules, and end products for membrane fatty acids, lipids, and sterols is known to mimic parts of fungal, bacterial-, plant-, and human lipid pathways, while completely duplicating none. Because leishmanial lipid metabolism is unique among organisms, genetically conserved (Wendt, et al., 1997, Science 277, 1811-1815), and biochemically-tightly regulated (Thompson, 1992, The Regulation of Membrane Lipid Metabolism. CRC Press, Ann Arbor, pp 230), the sterol pathway has the potential to provide us chemotherapeutic targets not duplicated in humans (drug development).
Leishmania share with plants (and animals) that they rely on mevalonic acid as a precursor for de novo sterol synthesis (Holz, 1985, Leishmaniasis. Elsevier, N.Y., pp 79-92; Thimann, 1977, Hormone Action in the Life of Plants. University of Massachusets press, Amherst, pp. 448; Thompson, 1992, supra) However, the major sterol of leishmanial and fungal membranes, synthesized de novo by these parasites, is not cholesterol (like humans), but a 24-substituted sterol (ergosterol or episterol or provitamin D2). Ergosterol is synthesized by these parasites de novo from acetylCoA, to mevalonate, to squalene, to lanosterol, and 4 steps later to ergosterol (Holz, 1985, supra). Coppens and Courtoy (1995, Mol Biochem Parasitol 73, 179-188) showed that procyclics of T. brucei normally contain ergosterol synthesized de nova, a pathway shared with Leishmania.
However, Leishmania require cholesterol. Unlike man, but like closely related Kinetoplastid parasites, of the genus Trypanosoma, Leishmania xe2x80x9csalvagexe2x80x9d cholesterol from their environment, i.e., from macrophages and monocytes (the LDL/cholesterol plasma clearance cells) in the mammalian reticuloendothelial system. Free cholesterol and free fatty acids do not occur normally in plasma. The cholesterol esters of fatty acids, which are by themselves insoluble in plasma, are located in the low density lipoprotein, LDL, as a nonpolar core surrounded with a polar shell of phospholipids, apoprotein, and unesterified cholesterol, thus ensuring solubilization and transport (Ormerod and Venkatesan, 1982, Microbiol Rev 46, 296-307; Thompson, 1992, supra). Leishmania reside in mononuclear macrophages, which comprise the major part of low-density lipoprotein (LDL) plasma clearance system via both receptor and receptor-independent mechanisms (Goldstein and Brown, 1976, Curr Top Cell Regul 11, 147-181; 1977, Ann Rev Biochem 46, 897-930; Weisgraber, et al., 1978, J Biol Chem 253, 9053-9062; Pangburn, et al., 1981, J Biol Chem 256, 3340-3347; Bilheimer, et al, 1982, Proc Natl Acad Sci USA 79, 3305-3309; Haughan, et al., 1992, Biochem Pharmacol 44, 2199-2206). Transport of LDL-cholesterol via either or both mechanisms into infected monocytes would thus allow leishmanial parasites to meet their cholesterol requirement. Drugs which interrupt the quantity, transport, or delivery of cholesterol to the parasite would have potential to adversely affect leishmanial survival.
There are marked metabolic similarities between leishmanial and trypanosomal lipid acquisition and metabolism. Bloodstream forms of Trypanosoma brucei spp. can ingest particulate fat (Wooten and Halsey, 1957, Parasitol 47, 427-431), and, like Leishmania, Trypanosoma brucei rhodesiense depends on the cholesterol of their habitat (Dixon et al., 1972, Comp Biochem Physiol 41B, 1-18).
Coppens and colleagues (1995, Mol Biochem Parasitol 73, 179-188) showed that the enzyme inhibitor, synvinolin (simvastatin or Zocor(copyright)), potentiates growth inhibition of Trypanosoma brucei in the presence of drugs interfering with the exogenous supply of cholesterol; and conversely, growth inhibition by synvinolin can be reversed by LDL, mevalonate, squalene or cholesterol. Coppens and Courtoy (1995, supra) showed that procyclics of T. brucei spp. normally incorporate exogenous cholesterol in their membranes. These investigators further demonstrated that growth of the culture-adapted trypanosomes is accelerated by supplementation of the medium with low density lipoprotein (LDL) particles which were endocytosed by the parasites via a receptor-mediated mechanism.
We observed that traditional medical herbal therapies, containing plant sterols having the cholestane backbone but with hydrophillic substitutent side chains, first destabilized then killed parasites in vitro in a dose-dependent manner. Chemical analyses of the structure of the antiparasitic active moieties from these plants ( greater than 70 tested) most frequently revealed an isoprenoid, terpenoid, or steroidal structure resembling but not duplicating normal mammalian sterolgenic precursors. It is known, as previously discussed, that Leishmania spp. and African Trypanosoma spp. take up cholesterol and any cholestane-backbone molecule (Dixon, et al., 1972, supra; Haughan, et al. 1995, supra). We believe that substitute xe2x80x9cplant cholesterol-likexe2x80x9d molecules serve to destabilize parasites"" membranes because of either addition of new hydrophillic sidegroups; or replacement of typically hydrophobic side-groups with more hydrophillic side-groups. These observations, in addition to the knowledge of the importance of cholesterol and cholesterol synthesis in the organism, appeared to validate the use of these medicinal plants as herbal remedies for treatment of protozoan parasitic infections.
Therefore, at several points within the sterol synthesis and cholesterol salvage pathways, we have identified molecules chemically or functionally similar to the natural component, but which act to shut-down leishmanial function.
Therefore, it is one object of the present invention to provide a novel method for identifying compounds having antiparasitic and antifungal activity based on the physiological action of the compounds in the sterol synthesis and/or metabolism, and/or excretion pathway of the parasite.
It is also an object of the present invention to provide a novel method for identifying antifungal and antiparasitic compounds by their ability to inhibit cholesterol synthesis and/or metabolism and/or excretion, directly or indirectly.
It is further an object of the present invention to provide novel antiparasitic and antifungal agents which are capable of oral administration, and are efficacious and less toxic alternatives to agents heretofore used for the treatment of fungal and/or parasitic infection in humans and animals.
A still further object of the present invention is to provide a novel method of using existing compounds not previously known to have antifungal or antiparasitic activity for the prevention and/or treatment of fungal or parasitic infection in humans and animals.
It is also an object of the present invention to provide antiparasitic and antifungal compositions for either prophylactic or field treatment.
A further object of the present invention includes the combined therapy that can be obtained by treating patients with leishmania, trichomoniasis, or trypanosomiasis, with a combination of the compounds of the present invention, preferbly the combination is chosen such that compounds which inhibit different parts of the cholesterol pathway are combined.